Clarified serum antitoxin and process of making the same



V in the methodof including Patented Nov. 27, 1923. f

* UNITED sures, 1"

ROBERT B. HENLEY, OE VIASHING Torr, Dreamer or ooLUDrBrA, DEDICATED, DY MESNE ASSIGNMENTS, 130 1333 PEOPLE, or 'rrrn UNITED STATES.

CL RIF D sn'ntriu an'rrrox ,No Drawing.

n AND B GE s r MAKINQ TEE Application filed June 21, 1922 Serial No. 570,008. I

(m n DTS H eel 0E M M e lat 2. STATE; a 2

To all whom it may concern; i

Be it known that I, RoBnRgrR'. HENLEY,

a citizen of the UnitedStates, and an employee of the United Agriculture, resid ng;

States Department of at. f WVashington, District of Columbia, have invented ac'ertain new and useful and Processes of Clarified Serum AntitoXin Mak ng the Same.

This application is made under the act of March 3, 1883, chapter 148,

St -a 1 and the, invention herein described and claimed may be used by the Government of the United States,

or any of its ofiicers or employees in the prosecution of Work for the Government, or any person 1n the United States, Without payment to me of any royalty thereon.

invention relates to an improvement separating serum from the corpuscles of defibrinatedblood, and the production by separation of the serum, by

heat or by filtration or .both, of

a clarified serum freed from all inert suspended matter corpuscles, debris, constituents and filterable and non-filterable micro-ora nisms.

This process is particularly applicable to the separation of a clarified serum antitoxin from old, phenolized, hog cholera defibrinated blood antitoirin, and the dethat will be given the separation of a scription of the process applies particularly to.

clear serum from old,phenolized hog cholera defibrinated blood antitoxin prepared as dis- 7 closed in Dorset Patent No. 823,110, but the process is-not limited in its application to this material, but may be clarified serum from any used to separate blood, either old or fresh, phenolized 0r non-phenoliz cd.

It is known that the protective principles and antibodies contained in'the blood from hogs which have been hyperimmunijz ed against hog cholera, as described by No. 823,110, r'eside exclusively in aten theserum of suchblood. At the present time these protective principlesare prepared and marbodies, or antitoxins,

keted in eitherronefof two forms:

the hyperi'mmunizecl hogs, is

blood from or anti- 1, The

draw ri t an ui bl Pre ervatives, such as phenol, added will be referred as s o jrin Dorset Patent No. 823,110. [This product o a pheee zed an i-h cholera defibrinated blood antitoXin; 2. The

second form of; antitoxin is the product D r t Y cells, which known as clear serum andis also covered e i P tent This cl er erum s W generally P ep r h v i non-phenolized defibrinated blood-antitoxin,

or fresh non phenoliaed citrated, or :o X'a-lated plasma, by the process disclosed by Dorset and Henley, U. S. Patent No. 1,26%,285.

This process described Dorset and Henley in U. S Patent No. 15,261,285, is limited in its application to fresh non phenolized defibrinated blood, or plasma and'ca nnfot b e applied to'old phenolized anti-hog -chol era defibri'nated blood antitoxin, to secure clear serum Which can be sterilized. A process that Will serve to'clarify old phenol ized anti-hog-cholera serum and at the same time give as, a product a clarified serum, which may be concentrated and eri z and 9 9' h s; W tl th em n simph l y and r p d t that. har ct r zes the.

separation of clear serum from' fresh defibrinated or citrated blood the Dorset V and Henley process, U. S. "Pat ent N0. 1,264,285, has been for a long time earnestlydesired by serum producers. "rhe'invefiaoa herein described Will fulfill this want the process possesses the same-rapidity and ease of operation that marks the method of in that a gas or fluid OflOW boiling point,

such as chloroform, is employed in. addition to the agglutinine and salt employed in the above process, and may in fact'be employed .without the presence of the agglutinine and salt. In fresh defibrinate-d blood antito rin the p are inert, areintact and are easily removed by the method of Dorsetand Henley. p the cells are in great part disruptedi'a'nd 111 old defibrinated blood antitoXi-n the cell contents, for the most. part hej moglobin, are distributed throughout the fluid. Neither agglutinine nor, salt will as i n the r meva if t e l 9 Y l e stituents from the disrupted cells, butf fl uids of low'boiling points suchas chloroform will combine with the disrupted cell contents, particularly hemoglobin, to form an insoluble compound which may be removed from the blood without injuring or otherwise adversely affecting the serum constitthe antibodies present in the original madefibrinated blood.

terial. The addition of'agglutinine or salt facilitates the rapid. removal of the cell debris and intact cells, andso are in practice ordinarily employed, but the presence of these materials is not an essentialfor the practical operation of the process.

In general the process is operated by adding to defibrinated blood, preferably phenol ized. a sufficient quantity of a chemical of low boiling point, to effect a precipitation of the hemoglobin. Agglutinine and salt added at this stage facilitate somewhat the later steps, but the addition of these materials is not essential.- Following the precipitation of the hemoglobin, the precipitated hemoglobin is removed by suitable mechanical means, affording as a product a clarified serum which contains all of the protective'antibodies present in the. original This clarified serum may be further purified by pasteurization, or filtration through bacteria proof filters, or both,and may be preserved by the addition of suitable quantities of acceptable preservatives.

The process as practicallyoperated con sists of four steps and the. description of each step follows:

Step Z.Addztion of precipz'tcmts.

tated by hand.

C. Addition of salts-Following completion of B5 gms. of common salt, sodium I V chloride, are added, and the flask and contentsjtransferred to a shaking machine.

.In this process B is an essential step. A and G are alternative steps and their employment usually facilitates the subsequent step of separation.

Step 2. S7;aking.

The material following completion of step 1 is transferred immediately to a shaking machine of any type-available and shaken until the blood becomes decidedly thick or by sedimentation,filtration, or centrifugali zation. In practise, either filtration or centrifugalization is employed. 7

' A. Centrifugalization-Any type centrifuge, continuous or bucket, may be employed and yields as product a clarified serum. which contains. the-antitoxin f and a densely packedclot which consists of inert cells, cell debris and cell constituents' This residue is discarded.

B. Filtration-The ,iclctted' bleed "is poured on a paper supported by a suitable funnel or other support. The filtrate'consists of the clarified theantibodies. The residue on the, paper mechanically retains some adherent serum,

and this is recovered by subjecting the residue to pressure in a suitable press. The pressings, after clarification by filtration, if necessary, are combined with I the clarified serum obtained by. direct filtration." The residue is discarded.

Step t.Fz'naZ procedure.

serum, which contains Yes The clarified serum separated this i is ready for use intreating hogs with hog cholera, or'it may be mixed with suitable antiseptics in order to preserve it, or it may be heated. to a temperature of 5860 C. for

one-half hour in order to destroy vegetative micro-organisms, or it may be ,passed through bacteria-proof filters which will remove all micro-organisms, living or dead, that may have survived the heating. In case heating is practised, the antiseptic is added subsequent to the heating, but the antiseptic may be added prior to or following filtration.

Having described my inventionl claim:

1. In the art of preparing a clarified serum from old phenolized defibrinated blood, the step comprising the addition to such blood of a chemical of low boiling point such as chloroform, and thereuponeffecting the separation of a a clarified serum "fromtlie clotted cell constituentsand dbrisloriigi nally contained blood. y V

2. Inthe art of preparing clarifiedho cholera serum anti-toxin from ;old, phenol ized, defibrinated 'blood hog cholera antitoxin, the step comprising the addition of chloroform to such antitoxin and thereupon effecting the separation of a clarified serum in the said. defibrinated antitoxin from the inert cell constituents and debris originally present in 'said defibrinated blood.

ROBERT R. HENLEY. 

